Protein-protein interactions are fundamental to proteomics (proteomics can be defined as the qualitative and quantitative studies of the proteome, the protein products of a species genome). Proteins are the most abundant and versatile macromolecules in living systems and serve crucial functions in essentially all biological processes. Proteins may perform structural, transport, protective, catalytic, sensory, neuro-transmitting, regulatory and many other functions. Though versatile as they are, considering the complexity of even the simplest life form, it is not surprising that proteins rarely function by themselves. Rather, they interact with other proteins and molecules. In the proteomic and genomic era, it has become clear to researchers that what constitutes a cell is the collective effort of these proteins whose functions are key to normal development and differentiation. Protein-protein interactions are fundamental to the understanding of biology, disease and even life itself.
In order to understand the normal biological processes and the diseases resulting from breakdown of normal functioning of proteins, it is important to study protein interactions. In the last twenty years science has made significant progress in the study of protein-protein interactions. The techniques developed include protein precipitation, transfection of suspected interacting proteins into host cells, in vitro biochemical analyses and yeast two-hybrid screening.
Thus far, the most versatile genetic system for screening and testing protein-protein interactions is the yeast two-hybrid (Y2H) system (Fields, S., and O. Song, “A novel genetic system to detect protein-protein interactions” Nature 340:245-246, 1989; U.S. Pat. Nos. 5,283,273, 5,468,614 and 5,667,973). Currently, it is estimated that there are at least 10,000 interactions among the 6,000 proteins in yeast (Uetz, P., “Two-hybrid arrays” Curr Opin Chem Biol 6:57-62, 2002). The actual number of protein-protein interactions is probably much higher, because the two proteins, bait and prey, studied by the Y2H method are not designed to be modified (see below) in any way. Thus, protein-protein interactions that require either interacting protein to be chemically modified will escape the detection by the Y2H method.
Post-translational modifications, or PTMs, refer to the specific chemical moieties added to target amino acid residues of proteins after the latter are synthesized (translated). Numerous proteins contain specific PTMs that are critical for their functions. PTMs may activate or inactivate the recipient proteins. Certain PTMs may flag the modified proteins for degradation or transport to selective intra- or extra-cellular destiny. Many more PTMs perform yet to be identified functions. Common PTMs include acetylation, phosphorylation, methylation, ubiquitylation, glycosylation, etc. Frequently, these PTMs are indispensable for the functions of the recipient proteins. However, in most cases, it is not known exactly what these PTMs do at a molecular level. One well-thought idea is that specific PTMs create new interface for protein-protein interactions. Some of these interactions may occur only after one of the two interacting partners is modified at a particular amino acid residue(s). For example, the well-conserved Phospho Tyrosine Binding (PTB) motif interacts with proteins that are phosphorylated at various tyrosine residues. The bromodomain that is shared by many transcriptional activators binds histones that are acetylated (Dhalluin, C., et al., “Structure and ligand of a histone acetyltransferase bromodomain” Nature 399:491-496, 1999; Jacobson, R. H., et al., “Structure and function of a human TAFII250 double bromodomain module” Science 288:1422-1425, 2000). In contrast, it is equally possible that existing protein-protein interactions may be inhibited if one of the two interacting proteins receives a particular modification. For example, the Silent Information Regulator protein Sir3 binds only to unacetylated histones for transcriptional repression (Edmondson, D. G., M. M. Smith, and S. Y. Roth, “Repression domain of the yeast global repressor Tup1 interacts directly with histones H3 and H4” Genes Dev 10:1247-1259). Acetylation of the histones antagonizes the function of Sir3 and leads to transcriptional de-silencing of the underlying genes (Carmen, et al., “Acetylation of the yeast histone H4 N terminus regulates its binding to heterochromatin protein SIR3” J Biol Chem 277:4778-81, 2002). These biochemical data support the idea that PTMs may positively or negatively regulate protein-protein interactions. However, these reports only represent sporadic examples of such regulation. In other words, to understand how PTMs regulate protein-protein interactions at a global and proteomic scale, a non-biased genetic method is needed.
In light of the primal importance of the possible effects of PTMs on protein-protein interactions, several groups independently reported a common strategy with which these authors were able to detect protein-protein interactions induced by specific phosphorylation (Cao, H., W. E. Courchesne, and C. C. Mastick, “A phosphotyrosine-dependent protein interaction screen reveals a role for phosphorylation of caveolin-1 on tyrosine 14: recruitment of C-terminal Src kinase” J Biol Chem 277:8771-8774, 2002; Shaywitz, A. J., S. L. Dove, M. E. Greenberg, and A. Hochschild, “Analysis of phosphorylation-dependent protein-protein interactions using a bacterial two-hybrid system” Sci STKE 2002:L11, 2002; Yamada, M., et al., “Analysis of tyrosine phosphorylation-dependent protein-protein interactions in TrkB-mediated intracellular signaling using modified yeast two-hybrid system” J Biochem (Tokyo) 130:157-65, 2001). In each case, kinases were expressed in the two-hybrid reporter cells that normally lack such enzyme systems. The bait proteins produced in these cells are thus modified by the foreign enzymes. For example, a tyrosine kinase and a serine/threonine kinase were expressed in yeast Shaywitz, A. J., S. L. Dove, M. E. Greenberg, and A. Hochschild, “Analysis of phosphorylation-dependent protein-protein interactions using a bacterial two-hybrid system” Sci STKE 2002:L11, 2002; Yamada, M., et al., “Analysis of tyrosine phosphorylation-dependent protein-protein interactions in TrkB-mediated intracellular signaling using modified yeast two-hybrid system” J Biochem (Tokyo) 130:157-65, 2001) and E. coli (Cao, H., W. E. Courchesne, and C. C. Mastick, “A phosphotyrosine-dependent protein interaction screen reveals a role for phosphorylation of caveolin-1 on tyrosine 14: recruitment of C-terminal Src kinase” J Biol Chem 277:8771-8774, 2002), respectively. The substrate proteins, existing in the form of two-hybrid baits, were shown to be phosphorylated in vivo and consequently allowed the detection of interactions involving protein phosphorylation Yamada, M., et al., “Analysis of tyrosine phosphorylation-dependent protein-protein interactions in TrkB-mediated intracellular signaling using modified yeast two-hybrid system” J Biochem (Tokyo) 130:157-65, 2001).
The above methods rely on the typical enzyme-substrate reactions occurring in trans (i.e., between two distinct proteins) to create the baits for genetic selection. Therefore, one concern is whether the efficiency of the bait modification would be sufficient to surpass the level of the unmodified bait, permitting a high signal-to-noise ratio in the genetic screen. To avoid this potential problem, one may choose to over-produce the foreign enzyme. However, such treatment may result in uncontrolled enzymatic action on host proteins, leading to cellular toxicity.
Unfortunately, there is not any method that enables researchers to screen for such interactions in a global, non-biased manner. Therefore, what is needed is an assay system that enables researchers to detect protein-protein interactions that are dependent upon or inhibited by post-translational modifications.